Molecular signature of stem-like glioma cells (SLGCs) from human glioblastoma and gliosarcoma

Glioblastoma multiforme (GBM) and the GBM variant gliosarcoma (GS) are among the tumors with the highest morbidity and mortality, providing only palliation. Stem-like glioma cells (SLGCs) are involved in tumor initiation, progression, therapy resistance, and relapse. The identification of general features of SLGCs could contribute to the development of more efficient therapies. Commercially available protein arrays were used to determine the cell surface signature of eight SLGC lines from GBMs, one SLGC line obtained from a xenotransplanted GBM-derived SLGC line, and three SLGC lines from GSs. By means of non-negative matrix factorization expression metaprofiles were calculated. Using the cophenetic correlation coefficient (CCC) five metaprofiles (MPs) were identified, which are characterized by specific combinations of 7–12 factors. Furthermore, the expression of several factors, that are associated with GBM prognosis, GBM subtypes, SLGC differentiation stages, or neural identity was evaluated. The investigation encompassed 24 distinct SLGC lines, four of which were derived from xenotransplanted SLGCs, and included the SLGC lines characterized by the metaprofiles. It turned out that all SLGC lines expressed the epidermal growth factor EGFR and EGFR ligands, often in the presence of additional receptor tyrosine kinases. Moreover, all SLGC lines displayed a neural signature and the IDH1 wildtype, but differed in their p53 and PTEN status. Pearson Correlation analysis identified a positive association between the pluripotency factor Sox2 and the expression of FABP7, Musashi, CD133, GFAP, but not with MGMT or Hif1α. Spherical growth, however, was positively correlated with high levels of Hif1α, CDK4, PTEN, and PDGFRβ, whereas correlations with stemness factors or MGMT (MGMT expression and promoter methylation) were low or missing. Factors highly expressed by all SLGC lines, irrespective of their degree of stemness and growth behavior, are Cathepsin-D, CD99, EMMPRIN/CD147, Intβ1, the Galectins 3 and 3b, and N-Cadherin.

Mouse model: processing of brains and stains : -In order to reveal and characterize orthotopic tumors, hematoxylin and eosin (HE) staining was performed according to standard protocols.-For this, mouse brains were cut in two halves at the puncture site, each half spanning both hemispheres.This was possible since the site of inoculation was visible as a small scar-like structure on the right hemisphere.-Starting from the two cutting surfaces, we sliced the mouse brains with a microtome.Up to 20 slices (4.5 µm) were taken from each side.Every fourth slice underwent HE staining; mounting was done with Entellan.-Microscopy was executed with the BZ8000 and the BZ9000 software.Microphotographs were analyzed for the presence of tumors.-In cases in which tumors were observed, we determined the cross-section dimension from the photos.Due to the irregular shape of most tumors and their invasiveness, it appeared inappropriate to provide exact measures.-In this context, it has to be noted, that tumors may undergo some shrinkage during the fixation and/or staining procedures.-Moreover, tumors may appear as structures with sharp boundaries in HE staining, though they are invasively growing.
Therefore, immunohistochemistry (IHC) was performed to test for invasiveness.IHC was carried out with a rabbit antibody against Sox2 and a mouse antibody specific for the human protein stem121.-IHC was also applied to reveal SLGCs in the background of more differentiated tumor cells.
-In the beginning we used three distinct concentrations of SLGC cells (50,000, 100,000, and 200,000).Since (i) we did not see any correlation between cell numbers and tumor size and (ii) only small tumors were generated with several SLGC lines, we decided to continue with 200,000 cells.-Please note: only data for tumors with known p53 status are shown in the figures and are listed in the tables.Supplementary: Expression of pten mRNA (RT-PCR) -PCR products were separated on 1.6% TBE agarose gels.
The samples -1 and -2 are biological replicates --T1439-F was grown in medium containing FCS. -PCR products were separated on 1.6 % TBE agarose gels.
Lower: Western blot analysis -10 % SDS-PAGE; transfer using semi-dry technique egfr/EGFR: epidermal growth factor receptor Supplementary: EGFR expression (Western blot analysis) -8% SDS PAGE.-SLGC lines (mc) and clones derived from the mc cultures by limited dilution assays were analyzed.-Amplification of truncated EGFR was observed for the T1464 mc and several T1464 clones, but disappeared over time in both.Supplementary: relative number of Integrin 6or v-positive cells in the cultures indicated (flow cytometry analysis).Two distinct biological replicates (I and II) were studied; cells were grown in N-medium.
Analyses with the Integrin 6or vantibodies were carried out in parallel.
relative number of CD133-positive cells in the cultures indicated and changes over progressing passages (flow cytometry analysis).